Introduction:

“Identifying the biological origin of a disease, and the potential targets for
intervention, is the first step in the discovery of a medicine.”

 A target is a broad term which can be applied to a range of biological entities which may
include for example proteins, genes and RNA.
 Target identification is the process of identifying the direct molecular target – for
example protein or nucleic acid – of a small molecule. In clinical pharmacology, target
identification is aimed at finding the efficacy target of a drug/pharmaceutical or other
xenobiotic.
 There is need to find a protein (e.g. receptor) or gene associated with a disease with
which a potential drug interacts – the so-called targets.

Note: Not every target is equally capable of affecting the course of a disease.
  The drug discovery process starts with the identification of a molecular target and the
next is the Target Validation. During target validation, its association with a specific
disease and its ability to regulate biological processes is tested in the body. The target
validation confirms that interactions with the target produce the desired change in the
behavior of diseased cells.
 It is the critical step in Drug discovery process. Identification of new drug targets, target
validation, biochemical assay development followed by LEAD identification provides
very important input in the development of new potential drug candidate.

Figure: steps of drug discovery

Characteristics of Drug Target

1. A specific drug target might have the following characteristics:

2. The drug target is a biomolecule(s), normally a protein that could exist in isolated or
complex modality.
 3. The biomolecules have special sites that match other molecules (commonly small
molecules with special structures). These molecules could be endogenous or extraneous
substances such as chemical molecules (drugs).
4. The bio-molecular structure might change when the biomolecule binds to small
molecules and the changes in structure normally are reversible.
5. Following the change in the biomolecule’s structure various physiological
responses occur and induce regulation of the cell, organ, tissue, or body status.
6. The physiological responses triggered by the changes in biomolecule structure play a
major role in complex regulation and have a therapeutic effect on pathological
conditions.
7. The expression, activity, and structure of the biomolecule might change over the
duration of the pathological process.
8. Small molecules binding to the biomolecules are drugs.

Traditional Drug Discovery v/s New Strategies in Drug discovery

 The path to identifying and validating the drug candidate molecule is not a one size- fits-
all-diseases strategy.
 The first stage in drug discovery process is to understand the disease mechanism, using
cellular and genetic approaches, in order to identify potential drug targets.
Role of target identification in drug discovery:

 Target identification and mechanism of action studies play an important role in small-
molecule discovery.
 A good target needs to be efficacious, safe, meet clinical and commercial needs and,
above all, be ‘druggable’. A ‘druggable’ target is accessible to the putative drug
molecule, be that a small molecule or larger biological and upon binding, elicit a
biological response which may be measured both in vitro and in vivo.
 Good target identification and validation enables increased confidence in the relationship
between target and disease and allows us to explore whether target modulation will lead
to mechanism-based side effects.

 Current therapy is based upon less than 500 molecular targets of about 10000 possible
targets:
a. 45% of which are G-protein coupled receptors
b. 28% are enzymes
c. 11% are hormones and factors
 d. 5% are ion channels
e. 2% are nuclear receptors

Hormones &
Enzymes factors Ion-
28% 11% Channels
5%

Nuclear
Receptors
2%

DNA
2%
Receptors
(GPCRs) Unknown
45% 7%

Figure: Current Drug Targets - few target classes; based on 483 drugs in Goodman and Gilman's "The Pharmacological
basis of therapeutics"
Source: Jürgen Drews, et al.Drug Discovery: A Historical Perspective. Science 287, 1960 (2000);DOI:
10.1126/science.287.5460.1960

 Target identification can be approached by direct biochemical methods, genetic

interactions, or computational inference. Combinations these approaches may be required
to fully characterize mechanisms of small-molecule action.
 Accordingly, the Broad Institute uses a multi-faceted approach to the target identification
problem in the context of genome-based drug discovery, including:
a. Quantitative proteomics based on mass spectrometry
b. Genetic complementation of small-molecule effects using RNA interference
c. Computational inference by connectivity analysis using reference compounds
 A forward genetics (or classical genetics) approach is characterized by identifying, often
under experimental selection pressure, a phenotype of interest, followed by identification
of the gene (or genes) responsible for the phenotype.
 Modern molecular biological methods, particularly genetic engineering approaches, have
given rise to reverse genetics (sometimes equated with molecular genetics), in which a
specific gene of interest is targeted for mutation, deletion or functional ablation (for
example, with RNAi11), followed by a broad search for the resulting phenotype.
 Figure. Mechanism-of-action and target identification in chemical genetics. (a) Target-based approaches
(reverse chemical genetics) begin with target validation, in which a role is established for a protein in a
pathway or disease, followed by a biochemical assay to find candidate small molecules; mechanism-of-
action studies are still required to validate cellular activities of candidates and evaluate possible side effects.
(b) Phenotype-based approaches (forward chemical genetics) begin with a phenotype in a model system
and an assay for small molecules that can perturb this phenotype; candidate small molecules must then
undergo target-identification and mechanism-of-action studies to determine the protein responsible for
phenotypic change.

Drugs Target at a molecular level:

 The main molecular targets for drugs are proteins (mainly enzymes, receptors, and
transport proteins) and nucleic acids (DNA and RNA).
 These are large molecules (macromolecules) that have molecular weights measured in the
order of several thousand atomic mass units. They are much bigger than the typical drug,
which has a molecular weight in the order of a few hundred atomic mass units. The
interaction of a drug with a macromolecular target involves a process known as binding.
 There is usually a specific area of the macromolecule where this takes place, known as
the binding site.
 Typically, this takes the form of a hollow or canyon on the surface of the macromolecule
allowing the drug to sink into the body of the larger molecule. Some drugs react with the
binding site and become permanently attached via a covalent bond.
 However, most drugs interact through weaker forms of interaction known as
intermolecular bonds .These include:
o Electrostatic or ionic bonds,
o Hydrogen bonds,
o Van der Waals interactions,
o Dipole–dipole interactions, and
o Hydrophobic interactions.

(It is also possible for these interactions to take place within a molecule, in which case they are
called intra molecular bonds.)

Note: None of these bonds is as strong as the covalent bonds that makeup the skeleton of a
molecule, and so they can be formed and then broken again. This means that, equilibrium
takes place between the drug being bound and unbound to its target.

 The binding forces are strong enough to hold the drug for a certain period of time to let it
have an effect on the target, but weak enough to allow the drug to depart once it has done
its job. The length of time the drug remains at its target will then depend on the number
of intermolecular bonds involved in holding it there.
 Drugs that have a large number of interactions are likely to remain bound longer than
those that have only a few. The relative strength of the different intermolecular binding
forces is also an important factor. Functional groups present in the drug can be important
in forming inter-molecular bonds with the target binding site. If they do so, they are
called binding groups.
 However, the carbon skeleton of the drug also plays an important role in binding the drug
to its target through van der Waals interactions. As far as the target binding site is
concerned, it too contains functional groups and carbon skeletons which can form
 intermolecular bonds with ‘visiting’ drugs. The specific regions where this takes place
are known as binding regions.
 The study of how drugs interact with their targets through binding interactions and
produce a pharmacological effect is known as pharmacodynamics.

Approaches to target identification:

There are three distinct and complementary approaches for discovering the protein target of a
small molecule:

1. Direct biochemical methods -

Direct methods involve labeling the protein or small molecule of interest,
incubation of the two populations and direct detection of binding, usually following some
type of wash procedure.

2. Genetic interaction methods –

Genetic manipulation can also be used to identify protein targets by modulating
presumed targets in cells, thereby changing small-molecule sensitivity.
Comparative genomics strategies aim to compare simultaneously two or more
genomes in order to identify similarities and differences, and hence identify potential
drug targets.
 3. Computational inference methods –
Target hypotheses, in contrast, can be generated by computational inference,
using pattern recognition to compare small-molecule effects to those of known reference
molecules or genetic perturbations.

Tools for target identification and validation:

 Reliable technologies for addressing target identification and validation are the
foundation of successful drug development. Microarrays have been well utilized in
genomics/proteomics approaches for gene/protein expression profiling and tissue/cell-
scale target validation.
 Antisense technologies (including RNA interference technology) enable sequence-based
gene knockdown at the RNA level. Zinc finger proteins are a DNA transcription-
targeting version of knockdown.
  Chemical genomics and proteomics are emerging tools for generating phenotype
changes, thus leading to target and hit identifications. Target identification with
proteomics is performed by comparing the protein expression levels in normal and
diseased tissues.
 NMR-based screening, as well as activity-based protein profiling, are trying to meet the
requirement of high-throughput target identification.

Microarrays:

 Target identification seeks to identify new targets, normally proteins (or DNA/RNA),
whose modulation might inhibit or reverse disease progression.
 Current technologies enable researchers to attempt to correlate changes in gene
(genomics) and protein (proteomics) expression with human disease, in the hope of
finding new targets.
 Assess gene and protein expression (via nucleic acid or protein microarrays) to identify
novel targets, and can also be used to validate the found targets at the tissue or cell scale
(via tissue or cell microarrays).

Nucleic acid microarrays

Today, nucleic-acid microarrays, which primarily use short oligonucleotides (15–25 nt), long
oligonucleotides (50–120 nt) and PCR-amplified cDNAs (100–3000 base pairs) as array
elements, are overwhelmingly dominant because of the relatively easy synthesis and the
chemical robustness of DNA.

Data generated from genome sequencing projects in several organisms has provided the
opportunity to build comprehensive maps of transcriptional regulation. Array-based gene
expression analysis (immobilized DNA probes hybridizing to RNA or cDNA targets) has
enabled parallel monitoring of cellular transcription at the level of the genome.

Thus, nucleic-acid microarrays have had a significant impact on our understanding of normal and
abnormal cell biochemistry and, thus, on the choice of targets for drug design.
In oncology, data generated from high density oligonucleotide microarrays from Affymetrix
containing 62 907 probe sets have been analyzed and compared, to identify 97 genes as
physiological targets of the retinoblastoma protein pathway, deregulation of which is a hallmark
of human cancer.

Further characterization of these genes should provide insights into how this pathway controls
proliferation, thus providing potential therapeutic targets.

Protein microarrays

Because most drug targets are proteins, protein and peptide microarrays are set to have an
important impact on drug discovery. Protein arrays, an emerging yet very promising technology,
are now being used to examine enzyme–substrate, DNA–protein and protein–protein
interactions.

By profiling the differential expression of proteins using antibody arrays and correlating the
changes to a disease phenotype, putative targets (and biomarkers) to a particular disease can be
identified, although to date, such microarrays have not been used to their full potential because
of difficulties with the technology.

This strategy is based on the mechanism of the reaction between the ligands and proteins, thus
demonstrating the approach as an activity-based and high-throughput method. Further
application of this method may lie on targeting known drugs or biological active compounds.

Tissue and cell microarrays

An alternative to the use of whole-tissue specimens is the use of live cell microarrays, which can
be used to identify potential drug targets by functionally characterizing large numbers of gene
products in cell-based assays.
Figure: Tools for target identification and validation.

Antisense technology:

A key strategy in target validation is to determine what happens, with respect to phenotype
and/or the expression of other genes in cells or model organisms, if a gene of interest is either
deleted or its activity is inhibited.

Gene knockout mimics the activity of a drug that completely inhibits the normal function of the
gene’s product.

Antisense oligonucleotides
Complementary to a portion of a target mRNA molecule, oligonucleotides are the original type
of molecule used for blocking protein synthesis of the target mRNA, and thus achieving the
knockdown of the target gene.

One example is the identification of COX17 as a therapeutic target for non-small cell lung cancer
(NSCLC).

RNA interference

RNA interference (RNAi) is another type of technology involving sequence-specific RNA for
use in gene knockdown.

This approach avoided some limits such as selectivity of mutational targeting, complexities of
anatomically based phenotypic analysis, or difficulties in subsequent gene identification, and
should make possible the systematic identification of components within each pathway, thus
leading to potential therapeutic targets.

Zinc finger proteins

Zinc finger proteins (ZFPs) have remarkable versatility for recognizing different sequences of
DNA, and variations in the amino acid sequence of the C2H2 domains allow them to be targeted
to different locations in the genome.

Each zinc finger is a short stretch of 30 amino acids, containing two conserved cysteines and two
conserved histidines. These proteins have been used as the DNA-binding domains of novel
transcription factors (ZFP TFs).

ZFP TFs can be applied to potential new drug target validation in two ways.

a. One direct way involves designing ZFPs to validate the phenotypic effects of
activating or repressing a gene.
b. Alternatively, libraries of ZFP TFs might be used to screen cells for desired phenotypic
changes.

Chemical genomics and proteomics

Rather than finding drugs for targets in the conventional pharmaceutical approach, forward
chemical genomics, in a sense, finds targets for known drugs.

Its goal is to discover the specific molecular targets and pathways that are modulated by
particular chemical molecules (i.e. study the biochemistry underling the phenotype changes
induced by chemicals).

Tagged library approach

Various kind of chemicals have been used to generate novel chemotypes and once an effect is
found, the next step is to identify the biological target using an affinity matrix made of the
immobilized hit compound.

High-throughput NMR-based screening is also utilized for fast identification of drug–target

interaction.

Affinity Chromatography as a Classical Method for Target Identification

Despite the large number of target identification techniques described to date, affinity
chromatography remains the most widely used method. The typical project begins with
structure–activity relationship (SAR) studies in which various functional groups of the small
molecule of interest are modified or removed to determine which one(s) are dispensable for drug
activity.

The primary limitation of affinity chromatography is the need to derivatize the small molecules
of interest. SAR studies are time-consuming and require extensive medicinal chemistry expertise
that is often lacking in the academic laboratories performing forward chemical genetics and
phenotypic small molecule screens.

A recent major advance in the affinity chromatography approach for target identification takes
advantage of the quantitative capability of SILAC to drastically increase the sensitivity of this
approach for target identification.

Drug affinity responsive target stability (DARTS)

DARTS is the method for target identification that relies on drug-induced protease resistance.
Drug affinity responsive target stability (DARTS) is a relatively quick and straightforward
approach to identify potential protein targets for small molecules. It relies on the protection
against proteolysis conferred on the target protein by interaction with a small molecule.

The basis for DARTS is that a protein becomes stabilized upon binding to a small molecule
compound or other ligand, which leads to decreased susceptibility of the target protein to
degradation by proteases. This decreased proteolysis is specific to the target protein(s) and
occurs for both high and low affinity compounds. Moreover, DARTS works especially well
using extremely complex protein samples such as whole cell lysates where non-specific protein-
ligand interactions are minimized due to the large number and variety of proteins in the mixture.
DARTS is advantageous because any small molecule can be used in its native form, meaning no
Structure-Activity Relationship (SAR) studies or chemical modifications to the ligand are
necessary for target identification.